Many organisms that inhabit environments where they are repeatedly exposed to freezing conditions have evolved specific antifreeze proteins (AFPs) that provide both freeze resistance and freeze tolerance (for review see Jia and Davies Trends in Biochemical Science 27:101–106, 2002). Significant potential exists to improve the survival of organisms, such as plants, by enhancing the expression of antifreeze proteins or by introducing antifreeze proteins into organisms that are currently freeze-intolerant. Such methods would, for example, extend the range of climates in which forage crops and other commercially useful plants could be produced. Such methodology could also be applied to microorganisms in order to increase survival during recovery from cold temperatures, or may be employed in a similar fashion to improve survivability associated with temperature fluctuations which occur during transportation at low temperatures.
Antifreeze proteins are found in a wide range of organisms including plants, fish, insects and bacteria. Although all AFPs are ice-binding proteins, there is a large variation in structure and amino acid sequence between organisms. For example, the AFPs from fish are very different from those of plants. Even within different plant species there is little amino acid sequence homology between known AFP proteins, indicating that AFPs have evolved separately in a number of plant species. This structural diversity has made it difficult to identify functional domains in the AFP proteins, as well as to identify residues involved in the ice-protein interaction.
It has been proposed that AFPs bind to ice as a receptor-ligand interaction, with ice and AFPs being the ligand and receptor, respectively, to prevent further growth of the crystals. In certain freeze tolerant organisms, such as plants, the ice forms in the extra-cellular space. At several degrees below the melting point there is a tendency for ice to re-crystallize, with large crystals forming at the expense of smaller ones. These large crystals have the ability to irreparably damage cells. The main role of AFPs is therefore to slow or halt this re-crystallization process. Plant AFPs in particular tend to be good at inhibiting the ice re-crystallization process thereby lessening the damage caused by freezing. This contrasts with AFPs of Antarctic marine fish, which have an additional thermal hysteresis (TH) activity that reduces the freezing temperature of water inside the fish by controlling ice crystal growth.
Plant AFPs have been isolated from rye, perennial ryegrass and carrot. The carrot protein contains leucine-rich repeats and has some similarity with polygalacturonase inhibitor proteins (Worrall et al., Science 282:115–117, 1998; Smallwood et al., Biochem. J. 340:385–391, 1999). Over-expression of the carrot antifreeze protein in transgenic tobacco plants resulted in accumulation of this AFP in the apoplast. In ice re-crystallization experiments, the carrot protein inhibited the size of ice crystals formed. The TH activity of the carrot AFP was low; between 0.2 to 0.6° C.
The perennial ryegrass AFP is rich in asparagine, valine and glycine residues and shares no sequence homology to the carrot AFP (WO 99/37782, Sidebottom et al., Nature 406:256, 2000). The grass AFP has ice re-crystallization activity similar to that observed for the carrot AFP and has a low TH (0.2–0.45° C.). A theoretical three-dimensional structure has been developed for the grass AFP protein showing that it has a β-roll conformation (Kuiper et al., Biophys. J. 81:3560–3565, 2001). This gives the protein a long flat structure and presents two large flat surfaces for ice-binding. The physico-chemical characteristics of an AFP derived from Lolium have recently been investigated (Pudney et al., Archiv. Biochem. Biophys. 410:238–245, 2003).
The scope for AFP applications extends from genetically modifying prokaryotic or eukaryotic organisms to produce formerly non-resident AFP proteins, into areas where AFPs are used as additives for cryoprotection. An example of this is molecular biology reagents such as restriction endonucleases, DNA modifying enzymes, DNA polymerases and associated buffers which are sensitive to freeze thaw. Molecular biology reagents which are particularly sensitive to freezing, such as in vitro transcription/translation systems could potentially benefit by the presence of AFPs. Whole cells, such as preparations of Escherichia coli, yeasts, blood platelets, red blood cells, ova and sperm, in addition to multicelluar complexes such as embryos and whole organs, could be protected by the ice restructuring properties of AFPs.
AFPs may also be usefully employed in frozen food products where small crystalline structure is desirable, such as ice cream, and to provide a superior food quality upon thawing of frozen food products such as frozen fruit. For example, International Patent Publication WO 92/22581 describes the use of plant AFPs in controlling ice crystal shape in ice cream. International Patent Publication WO 99/37782 describes the isolation of AFPs from grasses and the use of such AFPs in frozen food products, such as ice cream and frozen yogurt. A particularly attractive trait, which is exhibited by Lolium AFPs, is their stability at high temperature (Pudney et al., Archiv. Biochem. Biophys 410:238–245, 2003). This lends itself to applications within the food industry where high temperature treatments, such as pasteurization, are routinely used to inhibit microbial proliferation. AFPs may also be used in meat products to preserve texture and flavor after cold storage.
The ability to alter ice recrystallization may have wider applications within industrial crystallization processes. One example is separation, purification and consistency in the production of pharmaceuticals, agrochemicals and pigments. AFPs could also be employed in the sugar industry where controlling crystal formation is highly desirable.
Another area where the manipulation of crystal architecture is desirable is in healthcare. AFPs could be localized in tumours where their propensity to form hexagonal bipyramids would facilitate cellular damage. This type of treatment is particularly attractive because it is minimally invasive and does not have the accompanying negative side effects associated with traditional chemotherapy. Other healthcare applications include controlling the formation of biocrystals in disorders such as gout and in kidney stones.
It has been postulated that AFPs affect crystal formation by interfering with the molecular interactions between water molecules, see Jia and Davies, Trends in Biochemical Science 27:101–106, 2002. This could be used to assist the drying of, for example, dairy products or pharmaceuticals where a major component of process costs is incurred as part of the drying process.